Figure 6.

Anthocyanin regulates the effects of d‐gal on NO production and eNOS acetylation. HUVECs were treated with C‐3‐R, C‐3‐G, and mulberry extract, and then incubated with d‐gal for another 48 h. (a) NO release was measured as described in Materials and Methods. (b) Intracellular NO formation was measured via image analysis of NO‐sensitive fluorochrome, DAF‐2DA. (c) Quantification of DHR fluorescence intensity for peroxynitrite levels. (d) Nitrotyrosine and expression of β‐actin in HUVECs were determined by Western blotting. (e) Phosphorylation of eNOS at Ser1177 and expression of total eNOS and β‐actin in the HUVECs were determined by Western blotting. (f) SIRT1 and expression of β‐actin were determined by Western blotting. (g) NAD⁺ and NADH levels were measured, and NAD⁺/NADH ratios were quantified. Sirtuin activity. (h) Acetylated lysine and expression of β‐actin were determined by Western blotting. (i) eNOS was immunoprecipitated from HUVECs lysate, and its level of acetylation was analyzed by immunoblotting with anti‐acetyl‐lysine and anti‐SIRT1 antibody. Values are expressed as mean ± SE (n = 5, ^* p < 0.05 vs. control; ^# p < 0.05 vs. d‐galactose group)